In the pancreatic ȕ-cell, the GK protein levels show only marginal variations under physiological conditions, and the enzyme is considered to be regulated at the post- translational level [196]. However, post-translational regulation of GK in both ȕ-cells and hepatocytes is complex and only partially understood. It is established that GK activity, its subcellular localization and cellular stability are regulated by a spectrum of non-covalent GK-protein interactions that are different in E–cells and hepatocytes. In the hepatocytes, the interaction with the GK regulatory protein (GKRP) is a key short- term regulatory mechanism of GK activity [203, 204]. The 68 kDa GKRP, first discovered in rat liver, is an allosteric inhibitor of GK that binds preferentially to the super-open form of GK which predominates when glucose is low [15, 25, 203]. GKRP binds GK competitively with respect to glucose, and glucose binding releases GK from the GK-GKRP complex by inducing a conformational transition to the closed form. Physiologically, the GK-GKRP interaction and the subsequent inhibition of GK activity are promoted by fructose-6-phosphate, and suppressed by fructose-1- phosphate, compounds that bind to GKRP. Thus, the GK-GKRP interaction is modulated in response to fasting and feeding states [205, 206]. Importantly, GKRP provides a regulated translocation of GK between the cytosol and nucleus. As the glucose supply declines during periods of fasting, GKRP binds free cytoplasmic GK and transports it to the nucleus where GK is sequestered in an inactive state [207, 208]. Postprandially elevated glucose levels dissociate the GK-GKRP complex, and the active form of GK is translocated to the cytosol. This enables a rapid increase in GK activity and stimulation of glucose phosphorylation. Moreover, the GKRP-mediated compartmental redistribution of GK to the nucleus may serve to maintain a functional reserve of GK that can be quickly mobilized after a meal, in addition to stabilize and protect the enzyme from degradation by cytoplasmic proteolytic mechanisms [209, 210]. In GKRP deficient mice, the disruption of this regulation and the subsequent decrease in GK activity leads to altered glucose metabolism and impaired glycemic control [209]. Furthermore, functional studies on recombinant hGK enzymes have demonstrated that some GCK mutations cause a loss of regulation by GKRP which
may contribute to glucose intolerance in patients with GCK-MODY [65, 211-213]. The GKRP is not present or detectable in the pancreatic islets [214-216]. However, the presence of an inhibitory protein distinct from the liver GKRP has been suggested [215].
A second important regulator of GK activity is the bifunctional enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase (PFK2/FBPase2). The enzyme is expressed in both hepatocytes and E-cells where it is involved in the regulation of the glycolytic (E-cell and hepatocytes) and gluconeogenetic (hepatocytes) pathways [217]. PFK-2/FBPase-2 is a cytoplasmic binding partner of GK, and in insulin-producing cells this interaction has been demonstrated to enhance the catalytic activity of GK [217-219]. This effect may be, at least in part, due to stabilization of a catalytically favorable (closed) enzyme conformation at elevated glucose concentrations [218-220]. Beside its localization in the cytosol (hepatocytes and ȕ-cells) and in the nucleus in complex with GKRP (hepatocytes), GK has also been found to bind to subcellular structures such as mitochondria (hepatocytes and ȕ-cells) [221-223] and insulin- containing secretory granules (ȕ-cells) [216, 224, 225]. In mitochondria, GK is part of a regulatory multiprotein complex, and association of this complex with the outer mitochondrial membrane is dependent on the presence of the proapoptotic protein BAD [221, 223, 226]. Moreover, the phosphorylation status of BAD helps regulate the catalytic activity of GK. The detailed molecular basis for the GK/BAD interaction and its physiological significance for glucose metabolism, GSIS and apoptosis are, so far, not fully understood. Another potential mechanism for post-translational modulation of GK function, specific for the pancreatic ȕ-cell, arose from the observations that GK is part of the outer structure of insulin secretory granules in islet ȕ-cells and insulin- secreting ȕ-cell lines [216, 224, 225, 227, 228]. The granulelcytoplasm translocation of GK is regulated by insulin and, moreover, the release of GK from the granule- bound state was accompanied by an increase in enzyme activity [228]. Hence, it was suggested that changes in GK activity induced by association/dissociation from insulin granules may be implicated in the regulation of GSIS in pancreatic ȕ-cells [216].
Covalent post-translational modifications
Reversible post-translational modifications (PTMs) are used to dynamically modulate protein activity and stability. PTMs can occur at any step in the life-cycle of a protein serving various purposes, e.g. to mediate proper folding, cellular stability/turnover, subcellular localization, allosteric activation/inactivation, alter protein-protein interactions as well as to target a protein for degradation. There are > 200 different PTMs of which the majority occur by enzyme-mediated covalent attachment of a small chemical group, sugar, lipid or protein to one or more of the amino acid side chains in a target protein (e.g. glycosylation, acetylation, methylation, phosphorylation, S- nitrosylation, oxidation, ubiquitination, SUMOylation etc.). As already described, post-translational processes are important in the regulation of the cellular activity and stability of GK in hepatocytes and ȕ-cells, but the knowledge of covalent PTMs of GK in target cells and their possible regulatory functions has been very limited. However, studies on cultured ȕ-cells have demonstrated that the association of GK with insulin granules [216, 225] was dependent on its interaction with nitric oxide synthase (NOS) and that the localization and activity of GK was regulated by post-translational S- nitrosylation of the enzyme [216, 229]. Furthermore, it was suggested that defects in site-specific cysteine S-nitrosylation of GK are associated with naturally occurring GCK-MODY mutations in humans [230].
Ubiquitin (Ub) is a 76-amino acid globular protein (~8.5 kDa) that is highly conserved in eukaryotic cells. Ubiquitination (or ubiquitylation) is a reversible cellular process that involves the covalent attachment of one or several Ub proteins to a target protein [120, 231, 232]. Protein ubiquitination is an elegant example of how a single protein can regulate an array of diverse cellular processes such as cell cycle progression, regulated cell proliferation, cellular differentiation, apoptosis, transcriptional regulation and protein quality control (PQC) [233-236]. Given the central role of Ub in these processes, dysregulation of the ubiquitination machinery has been found associated (directly or indirectly) with the pathogenesis of many human diseases [237- 241]. In addition to Ub, there is a growing family of Ub-like proteins (UbLs) which,
similar to Ub, covalently modify proteins on lysines by related enzymatic pathways, but with distinct functional implications [242].
The ubiquitin conjugating system and ubiquitin-mediated proteolytic
pathway
Conjugation of Ub to a protein substrate or to itself usually involves the sequential action of three enzymatic reactions (Figure 9) [237, 243]. In the first step, Ub is activated for transfer by ubiquitin-activating enzyme E1 in an ATP-dependent reaction: E1 catalyzes the adenylation of Ub at the C-terminal glycine residue (G76), followed by the formation of an E1-S-Ub thioester intermediate [244]. In the second (ubiquitin-conjugating) step, activated Ub is transiently transferred from E1 onto an active site cysteine of the E2 enzyme via a trans(thio)esterification process to form another thioester bond [245]. To complete the enzymatic sequence, E2-S-Ub interacts with the E3 ubiquitin-protein ligase, which recognizes and associates with the substrate, promoting the transfer and conjugation of Ub [246, 247]. The Ub moiety is generally conjugated to target proteins through the formation of an isopeptide bond between a lysine (İ-amino group) on the protein and the C-terminal carboxyl group of Ub. The completion of one ubiquitination cycle results in a monoubiquitinated substrate. However, most often the cycle is repeated to form polyubiquitinated or multiubiquitinated substrates. The ubiquitin-chain is lengthened by the E3 ligase in collaboration with E1 and E2, sometimes with the help of an additional conjugating factor E4, specifically required for efficient multiubiquitin chain extension [248-250]. Eukaryotes are today estimated to have two E1 enzymes, ~40 E2 enzymes and > 600 E3s or E3 multiprotein complexes [251]. The E2-E3 pair is the primary determinant of substrate specificity [243]. Ubiquitination is reversible through the action of a large family of deubiquitinating enzymes (DUBs) (isopeptidases) which releases and disassembles polyubiquitin chains, enabling recycling of ubiquitin [252-254]. The human genome is predicted to encode ~95 DUBs which fall into at least five different classes [255].
Several ubiquitin-binding domains (UBDs), including UIM (Ub-interacting motifs) and UBA (Ub-association), can form transient, non-covalent interactions with either mono-ubiquitin or polyubiquitin chains [249, 250]. Cellular proteins containing one or several UBDs (often called ubiquitin receptors) are the immediate decoders of ubiquitination, being responsible for transmitting specific Ub signals to downstream cellular events. Importantly, the selective preferences of UBDs for ubiquitin chains of specific length and linkage are central to the versatile functions of Ub.
The topology of the polyubiquitin chain appears to be a function of specific E2s and the E2-E3 combinations involved [251-253]. All seven internal Lys residues (K6, K11, K27, K29, K33, K48 and K63) of Ub are believed to contribute to the assembly of polyubiquitin chains [254, 255]. This ability to form a variety of structures with diverse lengths and linkages distinguishes ubiquitination from UbL modification of proteins. It is generally assumed that the formation of polyubiquitin chains of different linkage types provides functional specificity that determines the fate of the modified protein [254].
Ub is best known for its function in targeting proteins for controlled degradation by the 26S proteasome [256, 257]. Most proteins degraded by the ubiquitin-proteasome pathway are linked to a polyUb chain in which the Ubs are coupled through isopeptide
Figure 9. The ubiquitin-proteasome degradation pathway. See text for details. The figure is reprinted from [248], with permission from Nature Publishing Group, a division of Macmillan Publishers Ltd © 2010.
linkages to K48 (or in some cases K11 or K29) on the preceding Ub. At least four sequentially added Ub moieties are believed to be required for efficient recognition and binding of the modified target protein to the proteasome [258]. In contrast, monoubiquitination and K63-linked polyubiquitin chains serve non-proteolytic functions in various intracellular pathways [234-236, 254, 259, 260]. Once a target protein is marked by K48-linked polyubiquitination, it appears to have a short half-life in cells, as it is rapidly degraded by the proteasome. Proteins can be targeted directly to the proteasome through recognition of polyubiquitin by the 26S complex or, alternatively, it can occur indirectly, mediated by various proteasomal shuttle factors [255]. The proteasome is a large (26S or ~2400 kDa) ATP-dependent multicatalytic protease that is present both in the nucleus and cytosol of eukaryotic cells [237, 261]. The 26S proteasome is composed of two subcomplexes, a 20S proteolytic core that provides the proteolytic activity needed to degrade modified substrates, and a 19S regulatory complex that confers the ability to recognize and unfold polyubiquitinated protein substrates and insert them into the proteolytic chamber [261]. The degradation through the proteasome is an irreversible process and the proteins are degraded to small peptides, most of which are hydrolyzed to amino acids by the sequential action of endo- and exopeptidases in the cytosol and nucleus. Proteins destined for degradation need to be deubiquitinated to ensure efficient substrate degradation as well as recycling of Ub, and tightly regulated deubiquitination is accomplished by proteasome-associated DUBs [262, 263]. The main steps in the ubiquitin-proteasome pathway are illustrated in Figure 9.
Role of the ubiquitin-proteasome pathway in ȕ–cell dysfunction and
hyperglycemia
Chronic hyperglycemia has been associated with oxidative stress and subsequent defective insulin secretion [264]. As previously mentioned, the pancreatic ȕ-cell is particularly vulnerable towards oxidative stress [179], which in turn may trigger the ubiquitin-proteasome pathway [265], suggesting a potential involvement of this pathway in hyperglycemia and ȕ-cell dysfunction [266]. Interestingly, the ubiquitin- proteasome degradation pathway has been ascribed a regulatory role in the pancreatic
ȕ-cell in glucose-stimulated (pro)-insulin synthesis, biogenesis and surface expression of the K+ATP channel and in maintaining the normal function of the Ca2+ channel – all essential components of GSIS [267-269]. However, little is known about covalent post-translational modifications of GK and their possible regulatory functions in the target cells, and the molecular and cellular mechanisms involved in the degradation/turnover of GK are also poorly understood. The possible implications of Ub (or UbLs) and the ubiquitin-proteasome pathway in this regard have not yet been subject for investigation and, hence, remain to be elucidated.